Targeted detoxification of selected reactive oxygen species in the vascular endothelium

Abstract

Oxidative stress underlies diverse vascular diseases, but its management remains elusive, in part because of our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites and our limited understanding which species need to be eliminated. The antioxidant enzymes (AOEs) superoxide dismutase (SOD) and catalase (which decompose and H(2)O(2), respectively), conjugated with an antibody to platelet-endothelial cell adhesion molecule-1 (PECAM-1), bind to endothelial cells and alleviate oxidative stress in cell culture models. Here, we studied the effects of these antioxidant conjugates in mouse models of vascular oxidative stress. Anti-PECAM/catalase and anti-PECAM/SOD conjugates, in contrast to control IgG/AOE conjugates, accumulated in the lungs and vascularized organs after intravenous injection in wild-type, but not PECAM KO mice. Anti-PECAM/catalase, but not anti-PECAM/SOD, protected mice from lung injury induced by H(2)O(2) produced by glucose oxidase deposited in the pulmonary vasculature. Anti-PECAM/catalase also reduced alveolar edema and attenuated decline in arterial oxygen in mice that underwent unilateral lung ischemia/reperfusion, whereas anti-PECAM/SOD was not effective, implying the key role of H(2)O(2) in tissue damage in this pathology. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase prevented oxidation of tetrahydrobiopterin and normalized vasoreactivity in the vessels of mice rendered hypertensive by pretreatment with angiotensin-II. This outcome agrees with reports implicating superoxide and peroxynitrite in altered endothelium-dependent vasodilatation in hypertension. Therefore, the use of endothelial cell-targeted antioxidants identifies the key specific species of ROS involved in various forms of vascular disease and holds promise for the mechanistically tailored treatment of these pathologies.

Fig. 1.

Vascular targeting of antioxidant enzymes conjugated with PECAM antibody. Distribution of ¹²⁵I-labeled SOD, catalase anti-PECAM, or IgG conjugates 1 h after intravenous injection in mice. A, ¹²⁵I-SOD conjugated with anti-PECAM () or control IgG (■). B, ¹²⁵I-catalase conjugated with anti-PECAM in the organs of wild-type C57BL/6 () or PECAM^−/− (■) mice. The data are shown as mean ± S.D, n = 3. *, P < 0.05 versus the corresponding control (■) group. WT, wild type; KO, knockout.

Fig. 2.

Anti-PECAM/catalase, but not anti-PECAM/SOD, attenuates acute lung injury and thrombosis in a mouse model of “double-hit” oxidative stress caused by H₂O₂ production and hyperoxia (“GOX”). Mice received intravenous injections of equimolar doses of anti-PECAM/catalase or anti-PECAM/SOD or a mixture of both conjugates. Ten minutes later, 0.75 μg/g i.v. anti-TM/GOX was injected and mice were placed in 80% O₂. Four hours later, mice were sacrificed, BALF obtained, and the lungs harvested and analyzed. A, degree of protection against lung edema tested by BALF protein level. B, attenuation of neutrophil alveolar transmigration and level of oxidative stress marker MDA in BALF (inset). *, P < 0.05 versus the corresponding control group. C and D, hematoxylin and eosin staining of lung tissue sections of GOX/hyperoxia challenged mice treated with saline (C) or anti-PECAM/catalase (D). Arrows in C show vessels with intravascular hemolysis and fibrin deposition (pink background), and arrows in D show congested vessels with intact red blood cells. E and F, autofluorescence of lung sections viewed via epifluorescence 4,6-diamidino-2-phenylindole/orange filter (380.0 nm/750.0 nm) show hemolyzed erythrocytes (arrow) in the vessels of PBS-treated mice challenged with GOX (E), and intact RBCs in anti-PECAM/catalase-treated mice challenged with GOX (F). G and H, anti-PECAM/catalase protects against pulmonary thrombosis caused by GOX/hyperoxia vascular oxidative stress. Blue color shows immunostaining for mouse fibrinogen. Cat, catalase; PMN, polymorphonuclear neutrophil.

Fig. 3.

Anti-PECAM/catalase, but not anti-PECAM/SOD, protects lungs against I/R. Mice were anesthetized and received intravenous injections of equimolar doses of catalase or SOD conjugates 30 min before left pulmonary artery clamping. Animals were subjected to ischemia (1 h) and then reperfusion (1 h) of the left lung. A, BAL proteins shown as percentage of protection versus sham-operated animals calculated as described in Materials and Methods. B, blood oxygenation in animals preinjected with conjugates. Upper and lower dash lines indicate blood oxygenation in sham and untreated I/R groups, respectively. #, P < 0.05, conjugate-treated group versus control I/R group; *, P < 0.05, anti-PECAM group versus control IgG group.

Fig. 4.

Anti-PECAM/SOD, but not anti-PECAM/catalase normalizes endothelial dysfunction induced by angiotensin II treatment. A and B, relaxation induced by acetylcholine was measured on preconstricted aortic rings obtained from mice from the indicated experimental groups: control untreated mice (○), AngII-treated mice (●), and AngII-treated mice received injections 15 min before aorta harvesting of anti-PECAM/catalase (▴ in A) or anti-PCEAM/SOD (■), or a mixture of unconjugated anti-PECAM and SOD (▴ in B). In C to E, aortas extracted from mice that underwent treatments as indicated have been analyzed for the level of: H₂O₂ (C), superoxide anion (D), and oxidized BH₄ (E). *, P < 0.05 versus control; #, P < 0.05 versus AngII, n = 4–9.